The emergence of antibiotic resistance has required that new approaches be applied in order to effectively fight a host of medically relevant bacterial infections. The limited group of antibiotics, currently in use, need to be replaced with novel, rigorous, and safe treatments in order to combat the evolved bacterium of today. One way to destroy bacteria is to target one of their most essential processes, metabolism. The recent discovery of RNA structural elements, termed riboswitches, that bind cellular metabolites and control expression of essential metabolic genes provides a unique and distinct target for development of artificial agonists to fight bacterial infections. Riboswitches are found in non-coding regions of messenger RNAs, and gene expression is modulated when metabolite binds directly to the RNA. Many riboswitches repress expression of nearby genes involved in the synthesis of the metabolite, providing an efficient feedback mechanism of genetic control. One particular riboswitch (the glmS riboswitch) binds to glucosamine-6-phosphate (GlcN6P), a building block of the cell wall in Gram-positive bacteria, and undergoes self-cleavage resulting in inactivity of the mRNA. The amine functionality of GlcN6P seems to be directly involved in RNA catalysis, whereas the phosphate may play a role in recognition of the ligand by the RNA. In order to develop effective artificial agonists/antibiotics that target the glmS riboswitch, an understanding of the structural and functional details of the riboswitch-metabolite complex is essential. The aims of this grant focus on (1) investigating the structural and catalytic roles of metal ions in the glmS riboswitch, (2) deciphering ligand recognition by the glmS riboswitch, and (3) designing non-natural agonists with the ability to stimulate glmS riboswitch self-cleavage and control gene expression. Using Nucleotide Analog Interference Mapping and Suppression (NAIM and NAIS, respectively) some of the long range contacts between the glmS riboswitch, its ligand, and metal ions will be determined. Using NAIM, the biochemical contribution of a single chemical group within the glmS riboswitch will be defined using nucleotide analogs that modify the atom(s) of interest. Using NAIS, specific tertiary hydrogen bonding partners within or involving the glmS RNA structure will be determined. Structure-function studies of riboswitches will enable rational design of non-natural metabolite-like compounds that might function as agonists/antibiotics to halt bacterial growth through alteration of gene expression. The threat of bacterial infections due to lack of effective antibiotics has come to the forefront as these pathogens become resistant to almost every antibiotic available to the public. The need is great for new classes of anti-microbial agents that target different, but specific and essential, metabolic pathways, such as those which utilize riboswitches to control gene expression. Structure-function studies of riboswitches will enable rational design of non-natural agonists that ultimately could function as antibiotics. [unreadable] [unreadable] [unreadable]